Process for DNA replication

ABSTRACT

A method is provided for replicating DNA, and in particular for replicating large segments of DNA. A primer is combined with a target DNA molecule to be replicated. The primer is designed to be at least partially homologous to a known site on the target DNA, and to create a D-loop when hybridized with that site. A replisome is then assembled at the D-loop, and this replisome creates a copy of the DNA, starting at the primer binding site. By utilizing two species of D-loop primers which bind to remote sites on the DNA flanking a region to be replicated, large sections of DNA can be replicated in a manner comparable to PCR. The replicated DNA can be analyzed to detect variations in the genetic sequence of the target, for linkage mapping and as a source of longer DNA molecules having a desired sequence.

This application is a §371 national stage application of InternationalPatent Application No. PCT/US00/04445 filed Feb. 3, 2000, and claims thebenefit of U.S. Provisional Application No. 60/118,703 filed Feb. 4,1999.

This application claims priority from U.S. Provisional Application No.60/118,703, which application is incorporated herein by reference forthose countries where such incorporation is allowed.

This application was supported by NIH Grant No. GM34557. The UnitedStates may have rights under this application.

BACKGROUND OF THE INVENTION

This application relates to a process for DNA replication, and to theapplication of this process for a variety of purposes.

Replication of DNA and other nucleic acids is a complex naturalphenomenon which occurs within all biological systems. To facilitate theexploitation of the resources represented in the diverse geneticmaterials of the world's organisms, however, it is desirable to be ableto replicate selected DNA sequences under more controlled conditions,for example to produce increased amounts of one sequence. Suchreplication of selected DNA sequences is required for a great manyapplications of potential scientific and industrial significance, andhas been accomplished by a variety of techniques. These include cloningof the DNA sequences into plasmids or genes, and replication of theplasmid using the DNA replication mechanisms of a host organism, andamplification techniques such as PCR or ligase amplification. Cloning iscapable of replicating complete gene sequences, but requires theintroduction of the sequences into a host organism, and the subsequentrecovery of the duplicated DNA. PCR and similar amplification techniquesoffer increased flexibility, including the ability to introduce labelsand/or sequence variations into the replicated DNA, and avoid the use ofa host organism, but are limited in the length of the sequence which canbe replicated. Thus, there remains a need for a methodology which willpermit the replication of long DNA molecules, while providing theflexibility associated with PCR amplification. It is an object of thepresent invention to provide such a methodology.

SUMMARY OF THE INVENTION

The present invention provides a method for replicating DNA, and inparticular for replicating large segments of DNA. In accordance with theinvention, a primer is combined with a target DNA molecule to bereplicated. The primer is designed to be at least partially homologousto a known site on the target DNA, and to create a D-loop whenhybridized with that site. A replisome is then assembled at the D-loop,and this replisome creates a copy of the DNA, starting at the primerbinding site. By utilizing two species of D-loop primers which bind toremote sites on the DNA flanking a region to be replicated, largesections of DNA can be replicated in a manner comparable to PCR.

The replicated DNA can be analyzed to detect variations in the geneticsequence of the target, for linkage mapping and as a source of longerDNA molecules having a desired sequence.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the scheme used for making a double-stranded circulartemplate DNA molecule containing a D-loop, which was used to validatethe concept of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for the controlled replication,generally in vitro, of selected regions of DNA. In accordance with theinvention, replication of a target region of a target DNA molecule isaccomplished by:

(a) introducing a D-loop into the target DNA molecule at a selectedinitiation point adjacent to the target region;

(b) assembling a replisome at the D-loop; and

(c) providing DNA monomers (dNTPs) and ATP, whereby the target region isreplicated. ATP is preferably provided at concentrations in excess ofabout 1 mM. ATP is required because the formation of a processive DNApolymerase complex requires ATP hydrolysis and also because DnaB, theDNA helicase, requires concentration in excess of 1 mM to be maximallyactive.

Introduction of a D-loop at a selected initiation site in duplex DNA canbe accomplished using an oligonucleotide primer which hybridizes withdouble-stranded DNA at a selected initiation site. The non-hybridizedstrand is displaced to create the D-loop. D-loop formation can be drivenby the homologous pairing enzyme, RecA, as has been described in theliterature. See, McEntee et al., Proc. Nat'l Acad. Sci. (USA) 76:2615-2619 (1979), which is incorporated herein by reference. D-loopformation could also be driven by other methods, for example heating ata moderately high temperature (for example 75-80° C.) may be enough todrive annealing, particularly in regions rich in A+T bases.

The oligonucleotide primer which is used for generation of the D-loopgenerally has a length of from 20 to about 50 bases. The primer isselected to be substantially complementary to one of the two strands ofthe target DNA duplex at the initiation site. As used herein, the term“substantially complementary” refers to a primer which will hybridizewith the target DNA duplex under conditions of moderately highstringency. However, it will be appreciated that RecA mediatedhybridization, if employed, is an enzymatic strand-pairing reaction, andthat conditions normally used for DNA-DNA hybridization (e.g. 0.6 MNaCl) would actually be inhibitory. Thus the precise conditionscorresponding to “moderately high stringency” may vary depending on themethodology used to drive the annealing. In a general sense, however,the term “substantially complementary” includes (1) primers which areperfectly complementary to the target DNA molecule, (2) primers whichare complementary for most of their length, but which include one orseveral mismatches from perfect complementarity, although not enoughmismatches to significantly reduce hybridization specificity; and (3)degenerate primers which include several bases at a given site toaccommodate a multiplicity of common alleles in the target DNA. The useof mismatched primers may result from the presence of a mutation in theinitiation site, or the mismatch may be intentionally selected forintroduction of a desired sequence variation into the replicated DNA.

The primers used in the invention may also include one or morenon-hybridized regions for the purpose of introducing a desiredadditional sequence into the replicated DNA. For example, thisadditional sequence may be a sequence which introduces a restrictionsite near the end of the replicated DNA to facilitate insertion of thereplicated copies into other DNA molecules. Preferred restriction siteswill be those recognized by rare-cutting restriction enzymes whichgenerally recognize 8-base sequences, or intron-homing endonucleasessuch as PI-SceI from yeast which recognizes a 31-base pair sequence.This will reduce the likelihood of cleavage occurring within thereplicated DNA at other than the intended cleavage site.

In an alternative embodiment of the invention useful withsingle-stranded templates, the primer used comprises a 3′- and a 5′region which are substantially complementary to portions of the targetDNA template, and a central non-complementary region which forms aD-loop when the primer is hybridized with the target DNA. A secondprimer which is complementary is used to form the invading strand of theD-loop. Similar variations for insertion of cleavage sites etc, may beincorporated in the structure of such primers.

The primers used in the method of the invention may also include adetectable label or capture moiety. Suitable detectable labels andcapture moieties are well known in the art as comparable materials areused in PCR, nucleic acid sequencing, and hybridization-based assays.Specific, non-limiting examples of suitable labels and/or capturemoieties include fluorescent dyes such as fluorescein, Texas Red orcyanine dyes; enzyme labels such as alkaline phosphatase; and capturablelabels such as biotin. Nucleic acid tails which specifically interactwith a known capture sequence can also be employed.

In a preferred embodiment of the invention, the primer is combined withtarget double-stranded DNA under conditions suitable for hybridizationand in the presence of the enzyme RecA, which results in the formationof a D-loop at the site of primer binding. Unlike common in vitroprocesses such as PCR, which utilize bacterial polymerases of inherentlylow processivity, the present invention utilizes replisome. Replisomesare multi-protein associations which form at a replication fork and actin concert to replicate DNA. Replisomes provide much greaterprocessivity than polymerases used for PCR. For example, the E. colireplisome can synthesize pieces of DNA at least as long as a megabase(1×10⁶ nucleotides). The fidelity of copying is also quite high, withthe E. coli replisome making fewer than 1 mistake in 10⁸ nucleotidessynthesized. Furthermore, unlike PCR, replisomes are substantiallyinsensitive to regions of secondary structure in the DNA template. Thus,utilization of replisomes offers numerous advantages over the use ofpolymerases.

Replisomes include proteins which perform a variety of functions.Replication of DNA using replisomes depends on an initial unwinding ofthe DNA duplex at an origin of replication, and the continued unwindingalong the strands as the replication process proceeds. This unwinding iscarried out by DNA helicases. The resultant regions of single-strandedDNA are stabilized by the binding of single-stranded DNA-bindingproteins which are also part of the replisome. The stabilizedsingle-stranded regions are then accessible to the enzymatic activitiesof polymerases enzymes required for replication to proceed.

Replisomes have been shown to be substantially self assembling. Thus,when the necessary proteins are present under appropriate conditions,the replisome will assemble. We have found that this assembly will occurat a D-loop. A preferred combination of a proteins for formation of areplisome in accordance with the present invention includes thefollowing proteins:

PriA, PriB, PriC, DnaT, DnaB, DnaC (primosomal proteins);

single-stranded DNA-binding protein (SSB); and

DNA polymerase III holoenzyme (Pol III HE).

An alternative combination utilizes the mutant protein DnaC810,(described below) in place of PriA, PriB, PriC and DnaT.

The preparation and recovery of these various proteins is well describedin the art, including the art cited below which is incorporated hereinby reference. Pol III HE may be used in a form recovered directly bypurification from E. coli, or as a combination of Pol III* and the βsubunit. Pol III HE may also be reconstituted from individuallyoverexpressed and purified subunits. These subunits are α (DnaE), ε(DnaQ), θ (HolE), β (DnaN), τ (DnaX, full length), γ (DnaX, truncated),δ (HolA), δ′ (HolB), χ (HolC) and ψ (HolD). Preparation of Pol III HE isdescribed in U.S. Pat. Nos. 5,668,004 and 5,583,026 which areincorporated herein by reference for those countries in which suchincorporation is permitted.

Replisomes have been found to initiate DNA replication at the site of aD-loop. Thus, the D-loop formed by the interaction of the primer withthe target DNA molecule serves as the initiation site for thereplication process in accordance with the invention. When appropriatenucleic acid monomers (i.e., deoxynucleotide triphosphates, dATP, dCTP,dGTP and dTTP) and ATP are available, a copy of the strand of the DNAmolecule to which the primer hybridizes is produced. The length ofreplicated material which can be produced in this way is much greaterthan the length which can be produced using PCR or comparabletechniques, with lengths in excess of 5000-500,000 bases being readilyattainable. Thus, the method provides the ability to make copies ofentire large genes, including both intron and exon sequences.

As will be apparent to persons skilled in the art, a person makingcopies of DNA will generally be interested in obtaining those copies ofa particular region of the DNA, which is referred to herein as the“target region.” The target region may be a particular gene, or aparticular portion of a gene depending on the use for which the copiedDNA is intended. The ability to produce copies of very large numbers ofbases changes the practical limits on the proximity between the primerand the target region from those which are usually observed in the PCRand comparable methods. Thus, while the initiation site must be“adjacent” to the target region, this means only that the initiationsite must be close enough to and on the correct side of the targetregion such that a replisome assembled at the D-loop will copy the DNAof the target region.

In a preferred embodiment of the invention, two primers are utilized.The first primer is as described above, and hybridizes with a firststrand of a double stranded DNA duplex. The second primer also is asubstantially complementary oligonucleotide primer, but it hybridizes tothe second strand of the DNA duplex at a second initiation site locatedon the other side of the target region. Thus, the two primers flank thetarget region, in the same manner that PCR primers flank a region to beamplified. Further, the same principle which leads to amplification ofjust the region bounded by PCR primers, leads to creation of much largerpieces of replicated DNA spanning the region between the two initiationsites using the method of the invention, although the efficiency may notbe as great as achieved with PCR. This reduced efficiency is less of aproblem than one might expect, however, since the large size of thereplicated DNA makes them inherently more detectable than smallfragments. On the other hand, since the process of the invention workson double-stranded DNA, it is not necessary to separate the strands ofthe target and the newly replicated DNA before proceeding with the nextcycle.

While the large size of the replicated DNA offers advantages forpurposes of detection, it may also pose problems. Very large DNAmolecules (i.e., those that are hundred of kilobases in length) arefragile, and nay be broken if manipulated in simple solutions. Thus,production of fragments of such lengths, and meaningful analysis of thelengths of such fragments may require that the reaction be performed ina supporting matrix, such as an agarose gel. Replicated DNA can betransferred out of the supporting matrix, for example for introductioninto a matrix for separation based on size by electrophoresis.

DNA replicated in accordance with the invention may be utilized for avariety of purposes. First, the replicated DNA may be used as a sourceof genetic material to be spliced into still larger nucleic acidconstructs, including plasmids, cosmids, viral vectors etc., tofacilitate expression of the replicated DNA in a suitable host system.Such splicing can be facilitated by the incorporation of restrictionsites near then ends of the replicated DNA as discussed above. When twoprimers are utilized, restriction sites can be introduced at both endsof the replicated DNA.

Second, the replication of DNA in accordance with this method can beused as part of a method for detecting genomic rearrangements in atarget DNA sequence. In such a method, a D-loop is introduced into theDNA at a selected initiation point, a replisome is assembled at theD-loop, and the DNA is copied to produce sufficient numbers of copiesfor analysis. The copied product is analyzed to detect variations insize or organization of the copied material using size-specificseparations, hybridization probes and other standard analyticaltechniques. It will be appreciated that the use of size-specificseparations requires the production of a product of defined lengths, andthus will generally require the use of the two primer embodimentdiscussed above. On the other hand, where the analysis involves themeasurement of the interaction of the DNA with a labeled or immobilizedprobe, the replication of multiple copies of a single strand of the DNA,without amplification, may be sufficient.

Third, the method can be used to facilitate linkage mapping. Forexample, the method can be used in the circumstance where twochromosomal markers are known to be near one another, but where theexact distance separating them is not known. D-loop oligonucleotideprimers are synthesized for each marker for both the DNA strands.Combinations of the pruners are used to replicate the region between thetwo markers, and the size of the product formed reflects the chromosomaldistance between the two markers. The method may also be used to mapunlinked genes, and markers such as RFLPs, SNIPs and ESTs.

To demonstrate the ability of the replisomes to assemble at a D loop andreplicate the DNA, we used a small bacteriophage DNA molecule as a modelsystem as described in the following non-limiting examples. Theconditions for replisome assembly and DNA replication can be extended touse with larger molecules, and with substantially complementary primersas discussed above.

EXAMPLE 1 Preparation of DNA Replication Proteins

To prepare DnaC810, a dnaC810 open reading frame was constructed bysplicing overlap extension polymerase chain reaction and cloned into theNdeI site of the pET11C overexpression plasmid (Novagen). Overexpressionand purification of DnaC810 was as for the wild type protein.

PriA, PriB, PriC, DnaT, DnaB and DnaC were purified by the methodsdescribed in Marians, K. J. Methods Enzymol. 262: 507-521 (1995). SSBwas purified using the procedures described in Minden and Marians, J.Biol. Chem. 260: 9316-9325 (1985). The DNA polymerase III holoenzyme waseither reconstituted from Pol III* and β subunit as described by Wu etal. J. Biol. Chem 267: 4030-4044 (1992) or from purified subunits asdescribed in Marians et al., J. Biol. Chem. 273: 2452-2457 (1998).

EXAMPLE 2

To validate the operability of the inventive concept, a double-strandedcircular template DNA was prepared in accordance with the steps shown inFIG. 1. A 100 nt-long oligonucleotide primer (Seq. ID No. 1) wasannealed to f1R408 viral DNA (Russell et al., Gene 45: 333-339 (1986)).The central 42 nt of this oligonucleotide are non-homnologous with thetemplate, thus forming a D-loop in the resulting heteroduplex.Incubation of the heteroduplex with DNA Polymerase III holoenzyme in thepresence of SSB and DNA monomers resulted in the extension of the primerand the formation of a nicked form II DNA with a 42 nt-long bubbleregion. During the last two minutes of this incubation, ddTTP and ddATPwere introduced at concentrations 20-fold higher than dTTP and dATP toensure that complementary strand synthesis could not be extendedfurther. After phenol extraction and ethanol precipitation, the DNAproducts were purified by electrophoresis through native agarose gels.Complete form II bubble DNA was recovered from the gel and a [5′-³²P]minus strand oligonucleotide (Seq. ID. No. 2) was then annealed to the Dloop form II template. The template was then gel filtered through BiogelA5M to remove unannealed oligonucleotide and unincorporated [γ-³²P] ATP.

EXAMPLE 3

Reaction mixtures (12 μl) containing 50 mM Hepes-KOH (pH 8.0), 10 mMMgOAc, 10 mM DTr, 80 mM KCL 200 μg/ml bovine serum albumin, 2 mM ATTP,40 ,μM dNTPs, 0.42 nM [³²P] form II D loop DNA template, 0.5 μM SSB, 225nM DnaC, 30 nM DNA polymerase III holoenzyme, PriA, PriB, PriC, DnaT andDnaB were incubated at 37 ° C. for 10 minutes. To test the sufficiencyof various combinations of proteins to replicate the template preparedin Example 2, reactions were also performed in which one of the proteins(PriA, PriB, PriC, DnaT, DnaC and DnaB) was omitted in each reactionmixture. As controls, template alone and template with the holoenzymealone were also evaluated. Reactions were terminated by the addition ofEDTA to a concentration of 25 mM and NaOH to a concentration of 50 mM.The reaction products were evaluated by electrophoresis at 2 V/cmn for20 hours at room temperature through horizontal 0.7% alkaline agarosegels using 30 mM NaOH, 2 mM EDTA as the electrophoresis buffer. The gelswere neutralized, dried and analyzed by autoradiography.

The electrophoresis gels showed that incubation of the D-loop template,the seven primosomal proteins, SSB and DNA polymerase III holoenzymeresulted in extension of the invading strand oligonucteotide (42 nt,Seq. ID. No. 2) to the full length template size (6.4 kb). Theefficiency of the reaction varied, but generally 15-30% of the invadingstrand could be elongated to full length in a 10 minute incubation. Thereaction exhibited an absolute requirement for all of the primosomalproteins except PriC. Omission of this protein resulted in a decrease inDNA synthesis to one-third that of the complete reaction. Thisobservation was similar to those reported for replication on differenttemplates. Ng et al., J. Biol. Chem. 271: 15642-15648 (1996). Someextension of the invading strand by the holoenzyme alone could beobserved, but this was suppressed by the presence of PriA. If theinvading strand was omitted from the reaction, and [α³²P] dATP wasincluded, no DNA replication could be observed.

EXAMPLE 4

Because DNA helicases were being introduced to the DNA during primroseassembly, extension of the invading strand could result from one of twoprocesses: either (1) assembly of a bona fide replication fork at the Dloop followed by elongation of the leading strand coupled with unwindingof the duplex DNA template, or (2) uncoupled unwinding of the templateDNA leaving an oligonucleotide annealed to the viral single stranded DNAthat could be elongated in a primer extension reaction by thepolymerase. We previously showed that coupled replication fork actionrequires a protein-protein interaction between DnaB and the τ subunit ofthe holoenzyme. Kim et al., Cell 84: 643-650 (1996). In the presence ofthis interaction, replication forks could move rapidly, at nearly 1000nt/sec, whereas in its absence, the polymerase becomes stuck behind aslow-moving helicase and replication fork progression proceeds at onlyabout 30 nt/sec.

To evaluate the mechanism active in the replication of DNA in the methodof the invention, the speed of elongation of the invading strand wasassessed in the presence and absence of τ using holoenzyme reconstitutedfrom individual purified subunits. Ten second time points were takenfrom the start of the reaction, and the elongated products were examinedon denaturing gels. Full length material could be observed in thepresence of τ after 10 seconds, whereas even after 60 seconds no fulllength material was observed in its absence. This corresponds to a rateof replication fork progression in the presence of τ of 600-700 nt/sec,similar to what has been observed in the past for other replicationsystems.

Mok et al., J. Biol. Chem. 262: 16644-16654 (1987). Thus, we concludethat bona fide replication fork assembly occurs at the D loop on thetemplate in the presence of primosomal proteins, SSB and the holoenzyme.

EXAMPLE 5

All of the phenotypes of priA null mutations can be suppressed bymutated priA alleles that encode PriA proteins that are no longerATPases or DNA helicases, but still catalyze primosome assembly. Zavitzet al., J. Biol. Chem 267: 6933-6940 (1992). These mutations aresubstitutions in the invariant Lys in the Walker A boxnucleotide-binding motif. If the PriA-dependent replication forkassembly described here were relevant to what happened in the cell, wewould expect these mutant proteins to substitute fully for wild-typePriA in the replication reaction. To test this, three mutant proteins,having the K230R, K230A and K230D substitutions were tested. All threesupported replication on the D loop to a greater extent than thewild-type protein. This same type of improved activity in the mutantproteins has been observed in other systems (Zavitz, supra), and mayarise because the mutant proteins remain bound to the site of DNAbinding, providing a better target than the wild-type protein that canmove off the site because of its helicase activity.

EXAMPLE 6

E. coli strains carrying priA mutations are very difficult to grow. Theyare rich-media sensitive, form huge filaments, and have a viabilityroughly one-hundredth that of the wild-type. Sandler et al., Genetics143: 5-13 (1996); Nurse et al., J. Bacteriol. 6686-6693 (1991); Masai etal., EMBO J. 13: 5338-5345 (1994). Suppressor mutations that restoreviability, as well as ablate constitutive induction of the SOS responseand the defects in homologous repair of UV-damaged DNA, arise overnightafter transduction of the priA2:kan allele into fresh recipient cells.The mutations map to dnaC. (Sandier, supra). DnaC forms a complex withDnaB in solution (Wicker et al., Proc. Natl. Acad Sci. (USA) 72: 921-925(1975), and is required for the efficient transfer of DnaB to DNA in thepresence of other replication protein. Marians et al., Ann. Rev.Biochem. 61: 673-719 (1992). In order to assess the biochemicalproperties of these altered DnaC proteins, one such suppressor allele,dnaC810, was molecularly cloned into an expression plasmid and themutant protein purified as described in Example 7, infra.

Strains carrying dnaC810 no longer require PriA for viability. Thissuggests that if the essential role for PriA in cellular metabolism wasto catalyze assembly of replication forks at recombinationintermediates, DnaC810 must be able to bypass the requirement for PriAto recognize the D loop and nucleate the assembly of a primosome.Accordingly, we tested whether DnaC810 alone could direct transfer ofDnaB to the D loop template DNA.

In the presence of SSB and the holoenzyme, the combination of wild-typeDnaC and DnaB did not support elongation of the invading strand of the Dloop. On the other hand, DnaC810 was clearly able to load DnaB to the Dloop on the template in the absence of the other primosomal proteins, asevidenced by the elongation of the invading strand to full length. Thus,the E176G substitution in DnaC810 represents a true gain of functionmutation that allows bypass of the DnaB loading pathway that involvesPriA, PriB, PriC and DnaT and permits a reduction in the number ofproteins necessary for the practice of the present invention.

Interestingly, the relative efficiencies of the replication reactioncatalyzed in the presence of DnaC810 and DnaB varied compared to thereaction catalyzed by the complete set of primosomal proteins. At 80 mMKCl, the DnaC810 reaction was 5- to 10-fold more efficient. However, at600 mM potassium glutamate, the reaction catalyzed by the complete setof proteins was more efficient by a factor of 2. While not intending tobe bound by a particular mechanism, this difference may arise fromdifferences in the relative stability of intermediate complexes that areformed during the loading of DnaB to DNA.

EXAMPLE 7 Construction of Plasmid pET11c-dnaC810

A dnaC810 open reading frame (ORF) was made by two-step overlappingpolymerase chain reaction (PCR) Morton et al., Gene 77: 61-68 (1989).The N-terminal coding region of dnaC810 was PCR amplified using plasmidpE11c-dnaC (Marians, K. J, Methods Enzynol. 262:m 507-521 (1995)) as atemplate and two flanking primers:

(i) the NdeI primer (Seq. ID No. 3), which carries a NdeI site at thednaC initiator codon, and

(ii) the AgeI′ primer (Seq. ID. No. 4), which carries the designed pointmutation (E176G, GAA-GGT). The C-terminal coding region of dnaC810 wasalso PCR amplified using plasmid pET11c-dnaC as a template and twodifferent flanking primers:

(i) the AgeI primer (Seq. ID No. 5), which is complementary to the AgeI′primer and

(ii) the BamHI primer (Seq. ID No. 6), which carries a BamHI site justdownstream of the dnaC stop codon. These overlapping N- and C-terminalfragments were gel purified after PCR and further PCR extended andamplified with the two flanking NdeI and BamHI primers. The gel purifieddnaC810 ORF fragment was digested with NedI and BamHI and ligated withNedI- and BamHl-digested pET11c plasmid DNA to give pET11 c-dnaC810.

Purification of DnaC810

Because of the extreme overproduction, DnaC810 was followed duringpurification by SDS-PAGE. BL21(DE3)pLysS carrying pET11c-dnaC810 wasgrown in 12 l L Broth (Mainatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1982)) containing 0.4% glucose and 300 mg/ml ampicillin to OD₆₀₀=0.4and then induced in the presence of 1 mM IPTG for 3 h. Cells werechilled, pelleted by centrifugation, and resuspended in 50 mM Tris-HCl(pH 8.4 at 4° C.) and 10% sucrose. The cell suspension (50 ml) wasadjusted to 150 mM KCl, 20 mM EDTA, 5 mM dithiothreitol, 0.02% lysozyme,and 0.1% Brij 58 and incubated at 0° C. for 10 min. This suspension wascentrifuged at 100,000×g for 1 h (Sorvall T865 rotor). The supernatant(fraction 1, 65 ml, 3510 mg protein) was adjusted to 0.04% polymin P bydropwise addition of a 1% solution. The precipitate was removed bycentrifugation at 47,000×g in a Sorvall SS-34 rotor for 30 min. Thesupernatant was further subjected to (NH₄)₂SO₄ fractionation (50%saturation) by the addition of solid. The resulting protein pellet wascollected by centrifugation at 47,000×g in a Sorvall SS-34 rotor for 30min. The protein pellet was resuspended in 8 ml of buffer A [50 mMTris-HCl (pH 7.5 at 4° C.), 1 mM EDTA, 5 mM dithiothreitol, 20%glycerol, 0.01% Brij 58] +50 mM NaCl to give fraction 2 (13 ml, 1108 mgprotein). Fraction 2 was dialyzed against 21 of buffer A+50 mM NaCl for12 h and then loaded onto a 100-ml DEAE-cellulose column (4 cm×20 cm)that had been equilibrated previously with buffer A+50 mM NaCl. Thecolumn was washed with 200 ml of buffer A+50 mM NaCl. Fractions (15 ml)of the flow-through and wash that contained protein were pooled to givefraction 3 (81 ml, 363 mg protein). Fraction 3 was loaded directly ontoa 35-ml SP-Sepharose FF column (formed in a 60-ml disposable syringe)that had been equilibrated previously with buffer A+50 mM NaCl. Thecolumn was washed with 200 ml of buffer A+50 mM NaCl and protein wasthen eluted with a 350-ml linear gradient of 50-300 mM NaCl in buffer A.DnaC810 eluted at 175 mM NaCl (fraction 4, 24 ml, 25 mg protein).Fraction 4 was then loaded directly onto a 6-ml hydroxylapatite column(packed in a 10-ml disposable syringe) that had been equilibratedpreviously with buffer A+200 mM NaCl. The column was washed with 12 mlof equilibration buffer and protein was eluted with a 60-ml lineargradient of 0-400 mM (NH₄)₂SO₄ in buffer A+200 mM NaCl. DnaC810 elutedat 150 mM (NH₄)₂SO₄ to give fraction 5 (5.2 ml, 16.5 mg protein).Fraction 5 was concentrated bydialyzing against buffer A+50 mM NaCl+30%polyethylene glycol 20,000 and loaded onto a 125-ml Superdex-200 FPLCcolumn that had been equilibrated with buffer A+50 mM NaCl. The columnwas eluted at 1 ml/min. Fractions (1 ml) containing DnaC810 were pooledto give fraction 6 (7.5 ml, 9.2 mg protein). Fraction 6 was then loadedonto a 3-ml phosphocellulose column that had been equilibrated withbuffer A+50 mM NaCl. The column was washed with 6 ml of equilibrationbuffer and protein was eluted with a 60-ml linear gradient of 50-400 mMNaCl in buffer A. DnaC810 eluted at 250 mM NaCl (Fraction 7, 3.5 ml, 5.2mg protein).

6 1 100 DNA Escherichia coli primer 1 acatacataa aggtggcaac gccattcgaaatgagctcca tatgctagct agggaggccc 60 ccgtcacaat caatagaaaa ttcatatggtttaccagcgc 100 2 42 DNA Escherichia coli minus strand oligonucleotide 2atataaaaga aacgcaaaga caccacggaa taagtttatt tt 42 3 34 DNA Escherichiacoli NdeI primer 3 taatgcaggc catatgaaaa acgttggcga cctg 34 4 24 DNAEscherichia coli AgeI′ primer 4 tcgtatttcg aaccggtctg cacg 24 5 24 DNAEscherichia coli AgeI primer 5 cgtgcagacc ggttcgaaat acga 24 6 37 DNAEscherichia coli BamHI primer 6 ttaagcactg ggatccttaa tactctttac ctgttac37

What is claimed is:
 1. A method for replication of a target region of atarget DNA molecule comprising the steps of: (a) introducing a D-Loopinto the target duplex DNA molecule at a first initiation point adjacentto the target region in a reaction mixture, wherein the step ofintroducing a D-loop is performed by hybridizing the duplex DNA moleculewith a first oligonucleotide primer which is substantially complementaryto the first initiation site; (b) adding proteins to the reactionmixture to assemble a replisome at the D-loop; and (c) providing DNAmonomers and ATP to the replisome, whereby the target region isreproduced, and further comprising the step of introducing a secondD-loop by hybridizing the duplex DNA molecule with a secondoligonucleotide primer which is substantially complementary to a secondinitiation site, said target region lying between the first and secondinitiation sites.
 2. The method of claim 1, wherein the firstoligonucleotide primer has a length of from 20 to 50 bases.
 3. Themethod of claim 1, wherein the first oligonucleotide primer comprises adetectable label or capture moiety.
 4. The method of claim 1, whereinthe first and second oligonucleotide primers each have a length of from20 to 50 bases.
 5. The method of claim 1, wherein at least one of theoligonucleotide primers comprises a detectable label or capture moiety.6. The method of claim 1, wherein the replication is performed in asupporting matrix.
 7. The method of claim 1, wherein the replisome isassembled via the action of primosomal proteins, single-strandedDNA-binding protein and the DNA polymerase III holoenzyme.
 8. The methodof claim 7, wherein the primosomal proteins includes a mutant PriAprotein which lacks ATPase and helicase functionality.
 9. The method ofclaim 3, wherein the replication is performed in a supporting matrix.10. The method of claim 3, wherein the replisome is assembled via theaction of primosomal proteins, single-strand binding protein andholoenzyme III.
 11. The method of claim 10, wherein the primosomalproteins includes a mutant PriA protein which lacks ATPase and helicasefunctionality.